Formulations of bacterial strains with insecticidal activity have been used for the control of insect pests in public health for more than two decade. For example, Bacillus thuringiensis subsp. israelensis has been used with remarkable success for the control of Simulium vectors in West Africa, resulting in the reduction of onchocerciasis, a disease of critical importance in that region. Similarly, this bacterium together with B. sphaericus has been used successfully against both Aedes and Culex in many developed and developing countries, thereby attenuating diseases, such as dengue and West Nile. However, the use of both these bacteria against Anopheles vectors of malaria has been much more limited, in part due to their lower activity against mosquitoes of this genus. In contrast Clostridium bifermentans subsp malaysia, another Gram positive spore forming strain isolated from Malaysia, has the highest toxicity to a number of Anopheles spp. Preliminary evidence supports our hypotheses, which are: a) that the mosquitocidal activity of this strain is due to toxins produced by the bacteria, and b) that toxins from this strain are novel. The focus of this R21proposal is therefore to identify the genes and isolate the toxic proteins using mosquito bioassays to drive both processes, and to characterize their activity against Anopheles, Aedes and Culex mosquitoes. This objective lays the ground work for our long-term objective of characterizing the molecular basis of selective activity of these toxins to different mosquito species. To achieve the objective proposed we will use both genomic and proteomic approaches. In the former approach, we have identified cosmid clones, prepared from genomic DNA, that are toxic to An. Stephensi larvae, and in this proposal we will characterize the critical gene(s) involved in toxicity using transposon mutagenesis and cosmid sequencing. As an additional approach we will purify the toxins involved using classical purification techniques, in conjunction with assays using larval mosquitoes, which will allow for monitoring purification of toxic proteins. Finally the toxins will be expressed to confirm their role in mosquitocidal toxicity. The proposed research will lead to the identification of new and novel mosquitocidal proteins. These protein toxins will provide additional tools that can be used to delay resistance development in mosquito vectors of human diseases and also, provide tools for optimization of mosquitocidal toxins that could be generated by modern biotechnological tools. [unreadable] [unreadable] [unreadable]